At the present time, the identity of an organism or infectious agent suspected of infecting a subject is normally determined by culturing a sample of biological material from the subject. For example, if it is suspected that a subject is suffering from an infection of the lung caused by the fungus Candida albicans, a sputum sample can be cultured. After a period of time, the culture is visually observed, and if a fungus grows in the culture in numbers sufficient to indicate a fungal infection, that fungus is identified by observing its morphological characteristics.
This method of confirming a diagnosis, however, has serious drawbacks. For example, it requires that the biological sample be cultured for a long enough period of time to allow a detectable amount of the organism to grow. This method also requires that the cultured sample be inspected by a technician trained in identifying different varieties of organisms. There is therefore a great need for an assay which can quickly and specifically identify an organism or infectious agent or a group of organisms or infectious agents. An assay which does not require a great deal of training to perform and interpret would also be advantageous.
There is also a need for an improved method for identifying other biological components present in a biological sample, where such components comprise polynucleotides or where the presence of such components is indicated by the presence of a polynucleotide. Present methods for detecting polynucleotides in a cell or tissue sample, such as the Norther blot method, require a relatively large amount of starting material. The Norther blot method is a widely accepted method of detecting specific genes (Sambrook J. et al., Molecular Cloning: A Laboratory Manual, 2nd ed., pp. 7.39-7.52) (hereinafter "Molecular Cloning"), and has been adapted for detecting cellular components such as jun oncogenes (Sherman, et al., Proc. Natl. Acac. Sci. USA, 87:5663-5666 (1990); Oursler M. J. et al., Proc. Natl. Acad. Sci. USA, 88:6613-6617 (1991)). In this method, mRNA is first purified from a tissue or cell culture, such as through electrophoresis on an agarose gel. Following electrophoresis, the mRNA is transferred onto membranes and hybridized with radioactive probes to identify positive band(s) through autoradiography. However, this method is not sensitive enough to identify signals corresponding to specific genes or gene products if the cells from which the mRNA is extracted have only a small quantity of genetic material.
Alternatively, reverse PCR (discussed in Molecular Cloning) can also be used to detect a wide variety of genes from different tissues and cells. In this method, mRNA is first converted to cDNA by reverse transcriptase, and specific gene fragments are then amplified by PCR using a set of primers (sense and anti-sense primers). The amplified gene can then be seen through agarose gel electrophoresis.